Groundwater contamination by organic pollutants is a subject of major concern in the United States and, increasingly, the rest of the industrialized world. The sources of pollution are almost as numerous as the types of pollutants found. Sources range from agricultural uses, injection wells, underground storage tanks and hazardous waste sites, to illegal dumping in localities with no prior history of toxic chemical exposure. In monitoring chemical waste disposal sites within the United States, pollutants have been categorized according to the frequency of detection of certain chemical classes and specific chemicals within that class. The priority pollutants are divided into volatiles, acid extractables, pesticides and base/neutrals. Volatiles are the most frequently detected group, owing largely to their widespread use as solvents. The most often encountered components of these volatile chemical pollutants in 1986 were, in order of frequency, trichloroethylene, tetrachloroethylene, trans-1, 2-dichloroethylene, 1,1-dichloroethylene, (ranked first, second, third and fifth respectively of all volatiles detected (Rajagopal, R. (1986) Environ. Prof. 8:244-264). The most prevalent of these, trichloroethylene (TCE), has been in use since the 1940's, and the subject of environmental and human health concerns since its discovery in a contaminated aquifer in 1979 (Muntner, J. E., S. P. Devries (1987) Toxics Law Reporter, Jan. 14:874).
All reports of TCE transformation by anaerobic bacteria indicate a very slow process. The rapid mineralization of TCE by aerobic bacteria has been demonstrated for several microbial isolates. All require the addition of exogenous chemicals to induce production of the requisite TCE degrading enzymes. Toluene serves to induce oxygenase enzymes of Pseudomonas putida F1 (Nelson, M. J. K., S. O. Montgomery, P. H. Pritchard (1988) App. Environ. Microbiol. 54:604-606) and P. cepacia G4 (Nelson, M. J. K., S. O. Montgomery, E. J. O'Neill, P. H. Pritchard (1986) Appl. Environ. Microbiol. 42:383-384). These enzymes are in turn responsible for TCE degradation by these bacteria. The route of toluene catabolism by P. cepacia G4 has been shown to proceed via a monooxygenation pathway that results first in an ortho-hydroxylation of toluene (catalyzed by o-toluene monooxygenase) and subsequently a second hydroxylation ortho to the first hydroxyl to form 3-methylcatechol (Shields, M.S., S.O. Montgomery, P. J. Chapman, S. M. Cuskey, P. H. Pritchard (1989) Appl. Environ. Microbiol. 55:1624-1629).
Several U.S. patents have issued concerning bioremediation, usually employing microorganisms in conjunction with exogenous chemicals as inducers. Microorganisms which require induction prior to gaining the capability of degrading certain compounds are, by definition, non-constitutive. Examples of these patents include U.S. Pat. Nos. 4,452,894; 4,477,570; 4,664,805; 4,713,343; 4,749,491; 4,853,334; 4,859,594; 4,925,802; and 4,954,258.
U.S. Pat. No. 4,452,894 concerns a pure culture of a Pseudomonas spp. that can utilize a variety of chlorinated aromatic chemicals as sole sources of carbon.
U.S. Pat. No. 4,477,570 concerns the isolation of bacterial strains, specifically Pseudornonas cepacia var. niagarous, that degrade aromatic and halogenated aromatic chemicals.
U.S. Pat. No. 4,664,805 concerns a method for degradation of halogenated organic pollutants through the addition of non-toxic chemical analogs with or without non-indigenous microorganisms.
U.S. Pat. No. 4,713,343 concerns bacteria which utilize lower alkane gases as carbon sources and which are induced to degrade certain chloroethanes and chloroethenes after exposure to lower alkane gases.
U.S. Pat. No. 4,749,491 concerns a method for stimulating indigenous bacteria to degrade chlorinated hydrocarbons through the addition of oxygen and propane or methane.
U.S. Pat. No. 4,853,334 concerns a loss of haloaliphatic hydrocarbons in the presence of a Pseudomonas fluorescens. However, the P. fluorescens, which is a completely different species than the subject bacteria, provide insufficient utilization of chloroaliphatics for purposes of bioremediation of contaminated environmental sites. P. fluorescens incubated with trichloroethylene (TCE) resulted in minimal loss of the TCE (approximately 2%) after 24 hours and resulted in a loss of less than 15% of the TCE after as much as five days.
U.S. Pat. No. 4,859,594 concerns degradation of certain organic chemicals by a variant of Pseudomonas cepacia. However, the P. cepacia described in this patent does not degrade trichloroethylene (TCE).
U.S. Pat. No. 4,925,802 concerns a method for stimulating biodegradation of halogenated aliphatic hydrocarbons. The method uses microbes and an inducer. Specifically exemplified is the non-constitutive microbe, Pseudornonas cepacia strain G4, which is the parent of the constitutive microbe strain of the subject invention.
U.S. Pat. No. 4,954,258 concerns the addition of alkanes or lower alkanols to methanotrophic bacteria for the degradation of TCE.
Current technology for the treatment of TCE-contaminated soil and water has relied primarily upon pump-and-treat systems whereby TCE is distilled away from the water under vacuum, or alternatively is air-stripped and transferred onto an adsorbent such as charcoal. Recent review articles on this subject strongly question whether this technology alone will ever be effective in the remediation of hazardous wastes like TCE, since even long term treatments have had only modest effects on pollutant concentrations (Travis, C. C., C. B. Doty (1990) Environ. Sci. Technol. 24:1464-1466). In either event, the result of currently employed treatments is simply the transfer of the pollutant to an adsorbent or to the atmosphere.
The capability to destroy the contaminant at the site represents significant environmental and economic benefits and fills the need for efficient bioremediation technologies. Major limitations to the bacterial systems described in the prior art for the bioremediation of TCE is that most degrade TCE fortuitously. That is to say, their ability to alter TCE is necessarily linked to the production of an enzyme that can accept TCE as a surrogate substrate, the native substrate being that which is used to induce the enzyme's synthesis. Due to this co-metabolic relationship, TCE cannot be degraded to any significant extent in the environment without the addition of an exogenous inducing substrate, because TCE does not itself induce the enzymes required for its own degradation. As a result, these prior art organisms are faced with the additional limitation of degrading TCE in the presence of the required cosubstrate that competes for the same active site on the induced enzyme. In addition, this also means that the organisms are not active beyond the environmental zone that can be controlled through the addition of effective concentrations of inducer. The active bacteria are effectively "tethered" to the inducing substrate. Both of these limitations have serious implications to the design of both environmental and bioreactor applications. In addition, the application of native inducing substrates such as toluene or phenol is not possible in the environment as they are themselves pollutants.
The use of the microbe of the subject invention, advantageously, does not have the problems associated with the use of prior art microbes to remediate sites contaminated with hazardous chemicals. Briefly, the microbe of the invention, P. cepacia strain PR1.sub.23 (formerly designated G4 5223 PR1 or G4 5223 Phe1), is a bacterial isolate that can function over a wide range of environmental conditions without the need for an added chemical inducer; it has a very high likelihood of competitive maintenance among native bacteria over the course of a given treatment; and it has evolved to utilize and grow on many organic pollutants likely to be found at waste treatment sites. One particular advantage is its efficient and rapid degradation of TCE.